This project is aimed at elucidating the mechanisms by which the Nef protein of primate immunodeficiency viruses (i.e., HIV-1, HIV-2 and SIV) downregulates CD4 in the viral host cells, T-lymphocytes and macrophages. Nef is a 27-34-kDa myristoylated accessory protein that is produced at high levels early during infection. Nef is an important determinant of pathogenicity, as demonstrated by the finding that some long-term non-progressors (i.e., infected patients who do not develop symptoms of AIDS for 10 years or longer) carry an HIV-1 strain with inactivating mutations of the Nef gene. An understanding of Nef function could thus provide new avenues for therapeutic intervention. Nef has many effects in the infected cells, the best characterized of which is the downregulation of CD4. Together with chemokine receptors, CD4 serves as a co-receptor for HIV-1 entry into cells. The downregulation of CD4 by Nef is thought to prevent superinfection and increase the release of infectious particles, thus explaining the higher virulence of Nef-carrying HIV-1 strains. [unreadable] [unreadable] Previous work from our laboratory demonstrated that the downregulation of CD4 by HIV-1 Nef requires clathrin and the heterotetrameric, clathrin-associated adaptor protein-2 (AP-2) complex in the host cells. In addition, we demonstrated that this requirement stemmed from a direct interaction of Nef with AP-2 involving a dileucine motif in Nef and the alpha and sigma2 subunits of AP-2. Since clathrin and AP-2 mediate endocytosis from the plasma membrane, these findings indicated that Nef downregulates CD4 by accelerating its internalization form the surface of host cells. This past year, we discovered that a second, diacidic motif in Nef interacts with a different binding site on the alpha-sigma2 hemicomplex. Interactions with both the dileucine and the diacidic motifs are required for Nef to downregulate CD4. [unreadable] [unreadable] Following Nef-induced internalization, CD4 is targeted for degradation in lysosomes, leading to a profound reduction of total CD4 levels in the cell. Little is known about how these late steps of CD4 downregulation are accomplished. The downregulation of some signaling receptors depends on ubiquitination-dependent targeting to the multivesicular body (MVB) pathway. We found that CD4 is ubiquitinated on all four lysine residues present in its cytosolic tail. However, Nef expression does not change the extent and pattern of CD4 ubiquitination. Moreover, in the presence of Nef, a CD4 mutant deficient in ubiquitination is internalized and degraded at rates similar to that of wild-type CD4. Nonetheless, Nef-induced CD4 downregulation is dependent on the MVB targeting machinery. These results indicate that Nef targets internalized CD4 for ubiquitination-independent delivery to the MVB pathway en route to lysosomes. [unreadable] [unreadable] Another effect of HIV-1 Nef on the host cells is the upregulation of immature class II molecules of the major histocompatibility complex (MHC-II) in association with the invariant chain (Ii). In collaboration with John Guatelli and colleagues (UCSD), we have found that this upregulation is caused by competition between dileucine signals in Nef and in the cytosolic tail of Ii for binding to the alpha-sigma2 hemicomplex. Overexpression of Nef during infection saturates the dileucine-binding site on AP-2, thus interfering with dileucine- and AP-2-mediated endocytosis of MHC-II-Ii from the cell surface. Trapping of MHC-II-Ii at the cell surface prevents MHC-II from acquiring viral peptides in endosomes, thus interfering with immune surveillance of infected cells.[unreadable] [unreadable] In collaboration with Eric Freed and colleagues (NCI), we have also examined the role of adaptor proteins in HIV-1 budding from the host cells. We found that the GGA proteins, which are monomeric adaptors associated with trans-Golgi network clathrin coats, modulate HIV-1 release into the extracellular space by acting on the small GTP-binding protein, Arf.